Use of phanquinone for the treatment of alzheimer&#39;s disease

ABSTRACT

The use of phanquinone for the manufacture of a pharmaceutical composition for the prevention or the treatment of Alzheimer&#39;s disease is disclosed. Also methods of treatment or prevention of Alzheimer&#39;s disease are disclosed.

TECHNICAL FIELD

The present invention relates to the use of a known compound for themanufacture of a pharmaceutical composition for treatment or preventionof Alzheimer's disease. Further, the invention relates to apharmaceutical composition for such treatment or prevention. Theinvention is also directed to methods of treatment or prevention ofAlzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD), which is the single major cause of dementia inadults in industrialized societies, is a degenerative brain disordercharacterized clinically by a progressive loss of memory, confusion,dementia and ultimately death. Histopathologically, Alzheimer's diseaseis characterized by the presence in the neocortex, especially thehippocampus, of two brain lesions: the neurofibrillary tangles (NFTs) ofpaired helical filaments (PHF) in the neurons and the neuritic (senile)plaques in the extracellular space. The formation of senile plaques isrelated to the appearance of the symptoms and signs of the disease,including amnesia. After the formation of senile plaque, neurofibrillarytangles are produced in the neuronal bodies. The formation ofneurofibrillary tangles is related to the worsening of amnesia and ofthe other symptoms of dementia.

A major component of the senile plaques is amyloid deposits. Cataract isa disease often occurring together with Alzheimer's disease. It islocated in the eyes and is also caused by amyloid deposits. Therefore,the treatment of patients having Alzheimer's disease disclosed in thepresent description and claims may equally be used to treat cataract. Asused in the present description and claims, the term Alzheimer'sdisease, therefore, also include the disease cataract.

A main component of the amyloid deposits is a polypeptide referred toherein as Aβ (Amyloid-beta). Aβ is normally a soluble component of thecerebrospinal fluid where it is found in concentrations of about 3-5 nm.Aβ may have 39 to 43 amino acids, typically 40 amino acids, in themature form and is derived as a proteolytic cleavage product from a cellsurface protein called the amyloid precursor protein (APP) (Kang et al.1987, Nature 325:733-736).

Many studies have shown that Aβ is toxic in vitro when added directly toneuronal cell cultures (Yankner B A, Duffy L K, Kirschner D A, Science1990, 250 (4978): 279-282; Koh J Y, Yang L L, Cotman C W, Brain Res1990, 533 (2): 315-320; and Pike C J, Burdick D, Walencewicz A J, GlabeC G, Cotman C W, J. Neurosci 1993, 13(4):1676-1687).

The neurotoxicity of Aβ has been located to be in the peptide sequencebetween amino acid residues 25 and 35 (Aβ(25-35)). Aβ(25-35) inducesneuronal cell death equally potent as full length Aβ(1-40) (Yankner B A,Duffy L K, Kirschner D A, Science 1990, 250 (4978): 279-282). The normalfunction of Aβ is not known at present but might be to form cationselective channels across cell membranes (Kawahara M. et al., 1997,Biophysical Journal 73/1, 67-75).

The precipitation of synthetic Aβ has been shown to be caused by severalenvironmental factors including low pH, high salt concentrations and thepresence of metals, e.g. zinc, copper, and mercury (Bush, A. I. et al.,1995, Science 268: 1921-1923). It has been reported that Aβ itselfspecifically and saturable binds zinc with a high affinity binding(K_(D)=107 nM) at a molar ratio of 1:1 (zinc:Aβ) (Bush, A. I. et al.,1994, J. Biol. Chem. 269: 12152-12158). This binding takes place atphysiological concentrations of zinc (Bush, A. I. et al., 1994, Science265: 1464-1467).

There is a strong supposition that removal of amyloid deposits frompatients suffering from Alzheimer's disease will alleviate the symptomsof Alzheimer's disease. Therefore, several attempts have been made toprepare such a drug, as methods for healing Alzheimer's disease areurgently sought.

International Patent Application, publication No. WO 93/10459, disclosesa method for the treatment of Alzheimer's disease by administering azinc binding agent. As preferred compounds, phytic acid,desferrioximine, sodium citrate, EDTA,1,2-diethyl-3-hydroxypyridine-4-one, and1-hydroxyethyl-3-hydroxy-2-methylpyridine-4-one are mentioned.

German patent application No. DE 39 32 338 discloses the use of analuminium chelator, such as 8-hydroxy-quinoline, for the treatment ofAlzheimer's disease.

U.S. Pat. No. 5,373,021 discloses the use of disulfiram and its saltsand analogs. According to this patent, the disclosed compounds may beused to reduce neurological damage caused by Alzheimer's disease.

International Patent Application, publication No. WO 98/06403 disclosesthe use of clioquinol for the manufacture of a pharmaceuticalcomposition for the treatment of Alzheimer's disease.

The hitherto known compounds suggested for the treatment of Alzheimer'sdisease have several drawbacks, which has prevented their widespreaduse. Most of the compounds are unable to penetrate theblood-brain-barrier and thus cannot readily reach the areas in which theamyloid is deposited. Disulfiram, which may penetrate theblood-brain-barrier, has the drawback that, when it is combined by apatient with ethyl alcohol, it causes severe adverse reactions,including headaches, nausea, vomiting, sweating, thirst, weakness, andlow blood pressure. Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline),which also may penetrate the blood-brain-barrier, has a damning historyas it as a side effect causes subacute myelo-optico-neuropathy (SMON).

Phanquinone (4,7-phenanthroline-5,6-dione) has hitherto been used forthe treatment of various disorders, such as amoebiasis. Phanquinone hasbeen sold by CIBA-GEIGY under the trademark ENTOBEX. In contrast toclioquinol no adverse side effects have been detected when phanquinoneis used in the normal dosage range.

In the past an antiamebic pharmaceutical preparation containing bothclioquinol and phanquinone has been sold by CIBA GEIGY under thetrademark Mexafor. However, the marketing of this preparation wasstopped when it was realized that clioquinol caused SMON.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a new use of a knowncompound for the treatment or prevention of Alzheimer's disease. Anotherobject of the present invention is to provide for an increased effect ofthe treatment of Alzheimer's disease. A further object is to avoid anydetrimental side effect of the treatment or prevention of Alzheimer'sdisease.

According to the present invention the use of phanquinone for themanufacture of a pharmaceutical composition for the treatment orprevention of Alzheimer's disease is provided.

Phanquinone may be administered in any amount efficient for thetreatment or pervention of Alzheimer's disease. Preferably, phanquinonemay be administered in an amount of 5 mg to 250 mg, and most preferred10 mg to 50 mg, one to three times daily.

In one embodiment of the invention, a compound, or a mixture ofcompounds, selected from the group comprising antioxidants,acetylcholine enhancers, trace metals, prosthetic groups and clioquinol,is administered prior to, together with or subsequent to theadministration of phanquinone.

The invention also relates to a pharmaceutical composition comprisingphanquinone and a compound, or a mixture of compounds, selected from thegroup comprising antioxidants, acetylcholine enhancers, trace metals,prosthetic groups and clioquinol provided, when clioquinol is selected,that at least one further compound is selected from the group.

The antioxidant is preferably vitamin C, vitamin E, Q10, or combinationsthereof. The acetylcholine enhancers are preferable μl agonists oranticholinesterase inhibibitors. Preferred anticholinesterase inhibitorsare tacrine (trademark: Cognex) and donepezil (trademark: Aricept). Theprosthetic group is preferably vitamin B₁₂.

In a preferred embodiment of the invention phanquinone and clioquinolare used for the manufacture of the pharmaceutical composition for thetreatment or prevention of Alzheimer's disease. Preferably, acombination of phanquinone, clioquinol and vitamin B₁₂ is used for themanufacture of the pharmaceutical composition.

The pharmaceutical composition may be formulated for oral, parenteral orintradermal administration. Further, the pharmaceutical formulation maybe formulated as a single pharmaceutical composition or as two or moreseparate pharmaceutical entities for sequential or substantiallysimultaneous administration.

The invention also relates to a method of treating a subject having orsuspected of having Alzheimer's disease comprising administering to thesubject an amount of phanquinone effective to treat or preventAlzheimer's disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the effect of phanquinone on Aβ(25-35) dose-response inPC12 cells.

FIG. 2 depicts the effect of phanquinone in Aβ(25-35) induced toxicityin PC12 cells.

FIG. 3 depicts the effect of phanquinone and clioquinol on Zn²⁺-inducedAβ aggregation.

FIG. 4 depicts the effect of clioquinol and phanquinone on Cu²⁺-inducedAβ aggregation.

FIG. 5 depicts the NMR spectra of three solutions. Solution a containedvitamin B₁₂ (hydroxycobalamin), at a concentration of 2.6 mM. Solution bcontained a mixture of 2.6 mM vitamin B₁₂ and 10 mM clioquinolglucuronide (mole ratio of about 1:4). Solution c contained 10 mMclioquinol glucuronide.

FIG. 6 depicts the right half of FIG. 5 expanded for easier comparisonof the resonance positions.

FIG. 7 depicts the left half of FIG. 5 expanded for easier comparison ofthe resonance positions.

DETAILED DESCRIPTION OF THE INVENTION

There is a need for introducing a drug on the market that is efficientin the therapy or the prophylaxis of Alzheimer's disease. According tothe present invention the known drug phanquinone is suggested.

In the examples it is shown that Aβ(25-35) in PC12 cells has apronounced effect on the redox activity of cells. In the method used inexample 2, the redox activity of cells exposed to 1 μM Aβ(25-35) isreduced to 60% of the normal level (see FIG. 2, open circles). However,when the Aβ(25-35)-treated cells is treated with 1 μg/ml phanquinone ormore the normal redox activity is restored. This indicate thatphanquinone when administered to a mammal such as a human will alleviatethe adverse effects of Aβ.

While it is not intended to limit the invention to any specificmechanism of action, it is at present believed that it is an antioxidanteffect of phanquinone that prevent the effect of Aβ on the cell redoxactivity.

The antioxidant effect of phanquinone may be improved byco-administering further antioxidant(s) such as vitamin C, vitamin E,vitamin Q10 or combinations thereof.

The presence of senile plaques in the extracellular space may prevent orinhibit the transmission of impulses in the cholinergic nervous system.The functioning of the nervous system is dependent on the mediatoracetylcholine. Therefore, compounds enhancing the level ofacetylcholine, such as compounds preventing or inhibiting the normalhydrolysis of acetylcholine by acetylcholinesterase, so-calledanticholinesterase inhibitors, may be administered together withphanquinone to improve the effect. Preferred anticholinesteraseinhibitors are tacrine and donepezil.

In a study reported in the examples, it is shown that phanquinone canreduce the aggregation of Aβ(1-40) induced by Cu²⁺ and Zn²⁺.

The following proposed mechanism of action of the invention is notintended to limit the invention to said mechanism. At present, applicantbelieves that phanquinone and Aβ competitively bind chelate zinc, copperand other heavy metals. Phanquinone has the ability to penetrate theblood-brain-barrier. When phanquinone has captured a heavy metal ion inthe extracellular space it moves into the blood and is cleared from thebody. As the aggregation or salt of Aβ and the heavy metal ion is inequilibrium with free Aβ and the free heavy metal ion phanquinone maycontinue to capture free heavy metal ions and move the heavy metal ionto the blood as long as aggregated Aβ, that is amyloid, is present. Thefree Aβ can not penetrate the blood-brain-barrier by passive diffusion,rather it will be degraded by the proteolytic enzymes normally presentin the extracellular space. Therefore, free Aβ or a degenerative partthereof may potentially damage the neuronal cells before it is digestedto a non-toxic extent. Meanwhile, phanquinone will prevent or inhibitthe potential cytotoxic effect of Aβ due to its ability to counteractthe reduction of the redox activity Aβ normally produce.

The reduction of the aggregation effected by phanquinone is 50-60% forCu²⁺ and 10% for Zn²⁺. It is further shown that clioquinol has theopposite tendency. Clioquinol reduces the Zn²⁺-induced aggregation ofAβ(1-40) by more than 60%, whereas the Cu²⁺-induced aggregation wasreduced by approximately 30%. In other words, phanquinone is best atreducing the Cu2+ induced aggregation of Aβ(1-40) and clioquinol is bestat reducing the Zn2+ induced aggregation.

As Cu²⁺ and Zn²⁺ naturally are present in the body of a patient havingor expected of having Alzheimer's disease, it may be desired to reduceor resolubilize the aggregation induced by both ions. Thus,administering of clioquinol prior to, together with or subsequent to theadministering of phanquinone may be preferred.

The administering of clioquinol is, however, problematic as clioquinolas a side effect causes SMON. Prior studies (see WO 98/06403) indicate apossible influence of clioquinol on vitamin B₁₂.

It is known that clioquinol is excreted through the kidneys asglucuronide or sulphate derivatives (Kotaki H., et al.: Enterohepaticcirculation of clioquinol in the rat, J. Pharmacobiodyn. 1984 June;7(6): 420-5 and Jurima M. et al.:Metabolism of14C-iodochlorhydroxyquinoline in the dog and the rat, J. Pharmacobiodyn.1984 March; 7(3): 164-70), e.g. as the compoundmethyl(5-chloro-7-iodo-quinolyl-2′,3′,4′-tri-O-acetyl-glucopyranosid)uronate.For short, this compound is referred to as clioquinol glucuronide in thefollowing.

The detoxification of hydrophobic substances, such as clioquinol, in thebody predominantly occurs in the liver. Therefore, it is believed thatthe clearance of clioquinol happens as follows: Clioquinol is convertedto clioquinol glucuronide in the liver. Following the formation, thewater soluble clioquinol glucuronide is secreted to the bile. The bileenters the intestine, wherein a major amount of the clioquinolglucuronide is evacuated in the stool. A certain amount of theclioquinol glucuronide is resorbed from the intestine to the blood. Theclioquinol glucuronide is filtered from the blood in the kidneys andappears in the terminal urine.

By treating mice with clioquinol and subsequently administering aradioisotope of vitamin B₁₂ ([⁵⁷Co]-cyanocobalamine) it is shown ininternational patent application, publication No. WO 98/06403 that theconcentration of vitamin B₁₂ in the brain and the liver of theclioquinol-treated mice remains at a normal level, whereas theconcentration of the radioisotope of vitamin B₁₂ is decreased in thekidney of such mice compared to the normal level. This finding suggestsa metabolism of vitamin B₁₂ being dependent on clioquinol. Further, thefinding suggests that the kidneys are the target organs, wherein theclioquinol dependent metabolism occurs.

In order to investigate a possible interaction between clioquinolglucuronide and vitamin B₁₂, an experiment was designed, whereinclioquinol glucuronide and vitamin B₁₂ were mixed in water. The mixturewas analyzed by ¹H NMR. The ¹H NMR spectra, see FIGS. 5-7, show thatsome of the resonances of vitamin B₁₂ (corresponding to thebenzimidazole moiety) have shifted, and the same is observed for tworesonances of the clioquinol glucuronide (corresponding to the quinolinemoiety). It is believed by applicant that similar results would beexpected using free clioquinol, however clioquinol cannot be dissolvedin aqueous solutions for NMR testing.

The results indicate a hydrophobic interaction between clioquinolglucuronide and vitamin B₁₂, possibly between the benzimidazole moietyof the vitamin B₁₂ and the quinoline moiety of clioquinol glucuronide.

Vitamin B₁₂ is normally resorbed actively from the renal plasma after ithas been filtered. In that way the body recovers most of the vitamin B₁₂that would otherwise have been lost in the urine. It has recently beendemonstrated that the resorption of vitamin B₁₂ is mediated by theaction of the membrane protein megalin (Moestrup S. K. et al. Proc.Natl. Acad. Sci. 1996; 93(16): 8612-7). The megalin is shown to have astrong affinity towards the binding of a complex formed by vitamin B₁₂and the transport protein transcobalamin.

Based on new finding reported herein, viz. that vitamin B₁₂ binds toclioquinol glucuronide, it is believed that vitamin B₁₂ does not bind tothe megalin protein and/or the transcobalamin when it is already boundto the clioquinol glucuronide. Thus, the resorption of vitamin B₁₂ willfail and the body will suffer from vitamin B₁₂ deficiency after acertain time period of clioquinol administration if the body is notsupplied with enough new vitamin B₁₂ through the normal diet.

The only source of vitamin B₁₂ input in man is food, since man cannotsynthetize it. Thus, diets low in meat and/or microorganisms willevidently cause vitamin B₁₂ deficiency. If persons are supplied withdiets with a too low content of vitamin B₁₂ the administration ofclioquinol will worsen the condition as the resorption of B₁₂ isprevented by the competitive binding of clioquinol with megalin. Thefact that SMON only was observed in Japan and that the Japanese dietpredominantly consisted of vegetables and cereals, especially rice,(Kromhout D, et al :“Food consumption pattern in the 1960s in sevencountries”, Am.J. Clin. Nutr. 49: 889-894, 1989) may explain why theSMON disease was confined to the Japan.

To counteract side effects of phanquinone and/or clioquinoladministration, it may be desired to co-administer trace metals and/orprosthetic groups. A preferred prosthetic group is vitamin B₁₂(cyanocobalamine).

By way of example, but not limitation, compositions, the use thereof,and methods of the invention can be indicated for: 1) patients diagnosedwith Alzheimer's disease at any clinical stage of the disease, 2) theprevention of Alzheimer's disease in patients with early or prodromalsymptoms or signs, and 3) the delay of the onset or evolution oraggravation of the symptoms and signs of Alzheimer's disease. Themethods and compositions of the invention will be, for example, usefulfor the treatment of Alzheimer's disease, the improvement or alleviationof any symptoms and signs of Alzheimer's disease, the improvement of anypathological or laboratory findings of Alzheimer's disease, the delay ofthe evolution of Alzheimer's disease, the delay of onset of anyAlzheimer's disease, symptoms and signs, the prevention of occurrence ofAlzheimer's disease, and the prevention of the onset of any of thesymptoms and signs of Alzheimer's disease or Parkinson's disease.

The subject, or patient, is an animal, e.g. a mammal, and is preferablyhuman, and can be a fetus, child, or adult.

The dose of phanquinone optimal in vivo for the resolubilization of Aβcan be determined by a physician upon conducting routine experiments. Anexample of such an experiment is the monitoring of soluble Aβ in thecerebrospinal fluid (CSF) (WO 93/10459, dated May 27, 1993 of Universityof Melbourne). Beginning with relatively low doses (10-25 mg/day), aphysician can monitor the amount of solubilized Aβ in the patient's CSF.If there is no increase in solubilized Aβ in response to the phanquinoneadministration, indicative of resolubilization of zinc-Aβ aggregates,the dosage can be raised until such an increase is observed. Anotherexample is monitoring the clinical signs and symptoms of the disease byusing clinical, behavioural and psychometric observations andmeasurements.

Prior to administration to humans, the efficacy is preferably shown inanimal models. Any animal model for Alzheimer's disease known in the artcan be used.

The pharmaceutical compositions according to the present inventionpreferably comprise one or more pharmaceutical acceptable carriers andthe active constituent(s). The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipients thereof. In a preferredembodiment, the phanquinone and optional further active constituents inthe pharmaceutical composition is purified.

It will be appreciated that the amount of phanquinone and optionalfurther active constituents required for said treatment or preventionwill vary according to the route of administration, the disorder to betreated, the condition, age, the file history of the subject, and thegalenic formulation of the pharmaceutical composition, etc. Whentreating a patient diagnosed as having Alzheimer's disease, the amountof phanquinone is preferably effective to increase the solubility ofAβ-aggregations in the cerebrospinal fluid of the patient.

In general, a suitable therapeutically effective amount of phanquinonein the pharmaceutical composition is, for example, 5 to 250 mg,preferably 10 to 50 mg. A suitable amount a compound, or a mixture ofcompounds, selected from the group comprising antioxidants, μ1 agonists,anticholinesterase inhibitors, trace metals, prosthetic groups andclioquinol in the pharmaceutical composition is, for example, 5 μg to250 mg, preferably 0.5 to 1 mg. If clioquinol and vitamin B₁₂ isselected, the amount of clioquinol preferably is effective to thetreatment or prevention of Alzheimer's disease and the amount of vitaminB₁₂ is preferably effective to inhibit a detrimental side effect ofclioquinol administration. The amounts of clioquinol and vitamin B₁₂ arepreferably 5 mg to 250 mg, most preferred 10 mg to 50 mg and 5μ to 2 mg,most preferred 0.5 mg to 1 mg, respectively.

The actually administered amounts of phanquinone and optional furtheractive constituents such as clioquinol and vitamin B₁₂ may be decided bya supervising physician. If the pharmaceutical composition in additionto phanquinone comprise further active constituents they can be in thesame composition for administering in combination concurrently, or indifferent compositions for administering substantially simultaneouslybut separately, or sequentially.

Therapeutic formulations include those suitable for parenteral(including intramuscular and intravenous), oral, rectal or intradermaladministration, although oral administration is the preferred route.Thus, the pharmaceutical composition may be formulated as tablets,pills, syrups, capsules, suppositories, formulations for transdermalapplication, powders, especially lyophilized powders for reconstitutionwith a carrier for intravenous administration, etc.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatine, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavouringagent, such as peppermint, methyl salicylate, or orange flavouring.

Therapeutic formulations suitable for oral administration, e.g. tabletsand pills, may be obtained by compression or moulding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bymixing the constituent(s), and compressing this mixture in a suitableapparatus into tablets having a suitable size. Prior to the mixing, thephanquinone may be mixed with a binder, a lubricant, an inert diluentand/or a disintegrating agent and the further optionally presentconstituents may be mixed with a diluent, a lubricant and/or asurfactant.

In a preferred embodiment, free-flowing phanquinone powder is mixed witha binder, such as microcrystalline cellulose, and a surfactant, such assodium lauryl sulphate, until a homogeneous mixture is obtained.Subsequently, another binder, such as polyvidone, is transferred to themixture under stirring. When a uniform distribution is obtained anaqueous solution of vitamin B₁₂ is added under constant stirring. Thismixture is passed through granulating sieves and dried by desiccationbefore compression into tablets in a standard compressing apparatus.

In a second preferred embodiment, free-flowing phanquinone powder ismixed with surfactants and/or emulsifying agents, such as Sapamine®(N-(4′-stearoyl amino phenyl)-trimethylammonium methyl sulphuric acid)and lactose monohydrate until a uniform distribution of the constituentsis obtained. A second preparation containing a disintegrating agent,such as maize starch, is added to the phanquinone mixture undercontinuous stirring. Such a second preparation may be prepared by addingexcess boiling water to a maize starch suspended in cold water. Thefinal mixture is granulated and dried as above and mixed with maizestarch and magnesium stearate and finally compressed into tablets in astandard apparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. Acoated tablet may be coated with sugar, shellac, film or other entericcoating agents.

Therapeutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of the active constituents. Anaqueous or oily carrier may be used. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soy bean oil,mineral oil, sesame oil and the like. Formulations for parenteraladministration also include a lyophilized powder comprising phanquinoneand optionally further active constituents that is to be reconstitutedby dissolving in a pharmaceutically acceptable carrier that dissolvesthe active constituents, e.g. an aqueous solution ofcarboxymethylcellulose and lauryl sulphate.

When the pharmaceutical composition is a capsule, it may contain aliquid carrier, such as a fatty oil, e.g. cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987);Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med.321: 574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23: 61 (1983); see also Levy et al., Science 228: 190 (1985); During etal., Ann. neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the central nervoussystem, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249: 1527-1533 (1990)).

In one embodiment of the pharmaceutical composition according to theinvention, phanquinone and the further active constituents, arecomprised as separate pharmaceutical entities. By way of example, oneentity may comprise phanquinone and clioquinol and another entity maycomprise vitamin B₁₂. The two entities, may be administeredsimultaneously or sequentially. For example, the entity comprisingphanquinone and clioquinol can be administered, followed by vitamin B₁₂administered within a day, week, or month of clioquinol and phanquinoneadministration. If the two entities are administered sequentially, theentity comprising phanquinone and clioquinol is preferably administeredfor one to three weeks followed by a wash out period of one to fourweeks during which the entity comprising vitamin B₁₂ is administered,but not the entity comprising clioquinol. After the wash out period, thetreatment can be repeated.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form described by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Other features and advantages of the invention will be apparent from thefollowing examples, which in conjunction with the accompanying drawingsillustrate by way of example the principles of the invention.

EXAMPLES Example 1

Effect of phanquinone on Aβ(25-35) dose-response in PC12 cells.

Aβ(25-35) was delivered by Bachem (CH) or Sigma (USA) and dissolved inphosphate buffered saline (PBS) at pH 7,4, 2 hours prior to application.The neurotoxicity of ARβ is located in the sequence between amino acidresidues 25 and 35 (Aβ(25-35)) and a decapeptide encompassing thisregion induces neural cell death equally potent as full length Aβ(1-40)(Yankner, Duffy L K, Kirschner D A: Neurotrophic and neurotoxic effectsof amyloid β protein: reversal by tachykinin neuropeptides. Science1990;250(4978):279-282).

Rat PC12 pheochromocytoma cells were grown in Dulbecco's modifiedEagle's medium (DMEM) containing 1% penicillin-streptomycin, 5% fetalcalf serum and 10% horse serum in humidified incubator with 5% CO₂.

PC12 cells were plated on 96-wells microtiter plates in 100 μl of theappropriate medium. After 24 hours the indicated concentrations ofAβ(25-35) peptide was added alone or together with phanquinone in thedesignated concentrations. Incubation continued for 24 hours. Followingincubation, MTT reduction was measured using a commercially availableassay according to the manufacturer's (Boehringer Mannheim)instructions. Assay values obtained by vehicle alone were defined as100%.

MTT is a substrate for intracellular and plasma membrane oxidoreductasesand has been widely used to measure reductions of cell redox activity.Reduction of the cell redox activity has been found to be an earlyindicator of Aβ mediated cell death (Shearman M S, Ragan C I, Iversen LL: Inhibition of PC12 cell redox activity is a specific, early indicatorof the mechanism of β-amyloid-mediated cell death, Proc. Natl. Acad.Sci. USA 1994;91(4):1470-1474).

To test the effect of phanquinone on Aβ(25-35) induced toxicity in PC12cells, PC12 cells were exposed to Aβ(25-35) peptide concentrationsranging from 0 to 10 μM. In the absence of phanquinone (i.e. onlyvehicle added) Aβ(25-35) produced a dose-dependent inhibition of MTTreduction (FIG. 1, filled-in circles). Concentrations of Aβ(25-35) aslow as 0.01 μM produced a significant reduction and at a concentrationof Aβ(25-35) or above 0.1 μM the MTT reduction was reduced to a maximumlevel of about 50%.

In the presence of 10 μg phanquinone per ml the toxic effect ofAβ(25-35) was virtually abolished (FIG. 1, open circles). Even atconcentrations of Aβ as high as 1 μM, the presence of phanquinonecompletely counteracted the toxic effect of Aβ. Only at the highestconcentration of Aβ(10 μM) there was a slight inhibition of MTTreduction by Aβ. This inhibition was, however moderate, only around 10%as compared to approximately 50% in the absence of phanquinone.

Example 2

Effect of phanquinone in Aβ(25-35) induced toxicity in PC12 cells.

In this study the same materials and methods as in example 1 were used,except that the PC12 cells were exposed to a fixed Aβ concentration of 1μM, whereas the concentration of phanquinone was varied between 0 and 10μg/ml. In the absence on phanquinone the Aβ(25-35) resulted inapproximately 40% inhibition of MTT reduction (FIG. 2, open circles).This inhibition was maintained in the presence of up to 0.1 μgphanquinone per ml. Increasing the concentration of phanquinone above0.1 μg/ml resulted in drastic reduced toxic effect of Aβ(25-35). Atconcentrations of phanquinone of 1 μg/ml and above, the toxic effect ofAβ(25-35) was completely abolished.

Example 3

Effect of phanquinone and clioquinol on Zn²⁺- and Cu²⁺-induced Aβaggregation.

A 5 mg/ml stock solution of Aβ(1-40) (delivered from Bachem (CH)) wasfreshly prepared before each experiment by dissolving the lyophilisedpeptide in 0.01 M HCl, followed by subsequent dilution 1:1 with 0.01 MNaOH to yield a neutral pH. Aliquot of Aβ(1-40) were diluted in PBS (pH7,4) to 100 μM and incubated at a total volume of 30 μl for 24 hours atroom temperature. For co-incubation experiments, the indicatedconcentrations of metal ions and/or aliquot of test compounds wereadded. The test compounds were added to a final molar concentration of10 μg/ml.

The amyloid formation was quantified by a thioflavin T fluorometricassay. Thioflavin binds specifically to amyloid and this introduces ashift in its emission spectrum and a fluorescent signal proportional tothe amount of amyloid is formed. After incubation, Aβ(1-40) peptideswere added to PBS (pH 6.0) and 3 μM thioflavin T in a final volume of 1ml. Fluorescence was monitored at excitation 454 nm and emission 482 nmusing a Fluoroscan II fluorometer (Molecular devices, UK). A time scanof fluorescence was performed and three values after the decay reached aplateau (around 5 minutes) were averaged after subtracting thebackground fluorescence of 3 μM thioflavin T. For co-incubationexperiments, fluorescence of test compound alone was determined. Sampleswere run in triplicate. The mean ±SD for the typical experiment is shownin the Figures (FIGS. 3 and 4).

Phanquinone and clioquinol were tested for their ability to prevent theaggregation of Aβ(1-40) into amyloid structures. Clioquinol is a knowncompound in the treatment of Alzheimer's disease (see internationalpublication number WO 98/06403) and was originally tested in the presentexperiment for comparison purposes.

It was studied whether the two compounds had any effect on metal-ioncatalysed Aβ aggregation, especially the aggregation caused by Zn²⁺ andCu²⁺. The results of the experiments are shown on FIGS. 3 and 4.

In the FIGS. 3 and 4 it is revealed that Cu²⁺ and Zn²⁺ to a lesserextent, increase the aggregation of Aβ into amyloid structures relativeto the spontaneous aggregation. In the presence of phanquinone andclioquinol at concentrations of 10 μg/ml, the metal ion inducedaggregation of Aβ was significantly reduced.

At the tested concentration of 10 μg/ml phanquinone reduced theCu²⁺-induced aggregation by 50-60%, while the Zn²⁺-induced aggregationwas only modest inhibited by approximately 10%. Unexpectedly, clioquinolshowed the opposite tendency. Clioquinol reduced the Zn²⁺-inducedaggregation of Aβ(1-40) by more than 60%, whereas the Cu²⁺-catalysedaggregation was reduced by approximately 30%. A pharmaceuticalcomposition comprising phanquinone in combination with clioquinol maythus have a more widely usage than a pharmaceutical compositioncomprising one of the compounds alone.

Example 4

In this example, a metabolite of clioquinol was synthesized.

It is known that the clioquinol is excreted through the kidneys asglucuronide derivatives of clioquinol (Kotaki H., et al.: “Enterohepaticcirculation of clioquinol in the rat”, J. Pharmacobiodyn. 1984 June;7(6): 420-5 and Jurima M. et al.:“Metabolism of14C-iodochlorhydroxyquinoline in the dog and the rat”, J.Pharmacobiodyn. 1984 March; 7(3): 164-70). The transformation ofclioquinol to the corresponding glucuronide presumable takes place inthe liver. Following the formation of clioquinol glucurunide in theliver it is eventually transferred to the kidneys for excretion in theurine.

A glucuronide derivative of clioquinol in the form ofmethyl(5-chloro-7-iodo-quinolyl-2′,3′,4′-tri-O-acetyl-glycopyranosid)uronatewas prepared according to the following reaction scheme:

A mixture of 5-chloro-8-hydroxy-7-iodo-quinoline (50 mg, 0.164 mmol),methyl 1-bromo-1-deoxy-2,3,4-tri-O-acetyl-D-glucopyranosiduronate (65mg, 0.164 mmol), CaSO₄.H₂O (35 mg) and pyridine (1.5 ml) was stirred atroom temperature for 20 min. Freshly prepared Ag₂CO₃ (35 mg) was addedto the reaction mixture and the suspended solution was stirred at roomtemperature for 20 hours in the dark. Subsequently, the reaction productwas deacetylated by 1 N aqueous NaOH.

The reaction mixture was diluted with CH₂Cl₂ (10 ml), filtered and thesolvent evaporated under reduced pressure. The above product waspurified by flash chromatography (TLC: CH₂Cl₂/MeOH 99/1, eluent:CH₂Cl₂/-MeOH 99.5/0.5).

NMR (400 MHz, CDCl₃) 2.04 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.13 (s, 3H,Ac), 3.68 (s, 3H, Me), 3.99 (d, 1H, 5′-H), 5.40-5.52 (m, 3H,2′,-3′,-4′-H), 6.29 (d, 1H, 1′-H), 7.56 (m, 1H, 3H), 7.99 (s, 1H, 6-H),8.52 (d, 1H, 4-H), 8.93 (s, 1H, 2-H).

This compound is referred to as clioquinol glucuronide in the following.

Example 5

The interaction of vitamin B₁₂ with clioquinol glucuronide as preparedin example 4, was studied using nuclear magnetic resonance (NMR)spectroscopy.

As the clioquinol glucuronide is soluble in water, the study wasundertaken in buffered water at pH=6.5. Three different solutions wereprepared and their ¹H NMR spectra were recorded in a DRX 400 MHzspectrophotometer at 20° C. Solution a) contained free vitamin B₁₂(hydroxycobalamin) in a concentration of 2.6 mM. Solution b) contained amixture of 2.6 mM vitamin B₁₂ and 10 mM clioquinol glucuronide (moleratio of about 1:4). Solution c) contained 10 mM clioquinol glucuronide.

In FIG. 5 the spectra of the three solutions are presented for thearomatic region (5.5-9.8 ppm). The differences are quite small butobvious in the expansion shown of FIG. 6 and FIG. 7, respectively. Someof the resonances of vitamin B₁₂ (corresponding to the benzimidazolemoiety) have shifted (see FIG. 6), and the same is observed for tworesonances of the clioquinol glucuronide (corresponding to the quinolinemoiety) (see FIG. 7).

The results suggest an interaction between clioquinol glucuronide andvitamin B₁₂, possibly of a hydrophobic nature between the benzimidazolemoiety of the vitamin B₁₂ and the quinoline moiety of clioquinolglucuronide.

The hydrophobic binding of vitamin B₁₂ to clioquinol glucuronide isbelieved to cause the vitamin B₁₂ to be excreted from the body togetherwith clioquinol glucuronide, thus preventing resorption of vitamin B₁₂,which would eventually lead to a vitamin B₁₂ deficiency. Therefore,vitamin B₁₂ deficiency is believed to be, at least to some extent, theunderlying cause of SMON. In consequence, whenever clioquinol isadministered it should be ensured that the level of vitamin B₁₂ in thetreated subject is sufficient for avoiding deficiency. This may beaccomplished by co-administering of clioquinol and vitamin B₁₂.

Example 6

Preparation of a pharmaceutical composition comprising phanquinone.

250 g of phanquinone was mixed with 200 g sapamine® (N-(4′-stearoylamino-phenyl)-trimethylammonium methyl sulphuric acid) and 1025 glactose mono-hydrate for a period of 5 minutes. 300 g of boiling waterwas added in one go to a mixture of 100 g maize starch in 100 g coldwater. The maize suspension, cooled to 40° C. was added to thephanquinone containing powder mixture under continuous stirring. Themixture was granulated using a 2.5 mm sieve and desiccated for 18 hoursat 40° C. The dry granules were mixed with 400 g maize starch and 20 gmagnesium stearate. The final mixture was formulated into tablets havinga diameter of 8.0 mm and a weight of 200 mg.

Example 7

Preparation of a pharmaceutical composition comprising phanquinone,clioquinol and vitamin B₁₂.

250 g phanquinone and 250 g of clioquinol was mixed with 200 g sapamine®(N-(4′-stearoyl amino-phenyl)-trimethylammonium methyl sulphuric acid)and 1025 g lactose mono-hydrate for a period of 5 minutes. 300 g ofboiling water was added in one go to a mixture of 100 g maize starch in100 g cold water. The maize suspension, cooled to 40° C. was added tothe phanquinone and clioquinol containing powder mixture undercontinuous stirring. Subsequently, an aqueous solution of 5 g vitaminB₁₂ was added. The mixture was granulated using a 2.5 mm sieve anddesiccated for 18 hours at 40° C. The dry granules were mixed with 400 gmaize starch and 20 g magnesium stearate. The final mixture wasformulated into tablets having a diameter of 8.0 mm and a weight of 200mg.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variation are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications, as would be obvious to a person skilled in the art, areintended to be included in the scope of the following claims.

What is claimed is:
 1. A method of treating a subject having or suspected of having Alzheimer's disease comprising administering to the subject an amount of phanquinone effective for the treatment or prevention of Alzheimer's disease, said subject being in need of said treatment or prevention.
 2. The method of claim 1 comprising administering to the subject an amount of phanquinone effective to increase the solubility of amyloid-beta (Aβ) in the cerebrospinal fluid of said subject.
 3. The method of claim 1, further comprising administering a compound or a mixture of compounds selected from the group consisting of antioxidants, acetylcholine enhancers, trace metals, and prosthetic groups.
 4. The method according to claim 3, wherein the total amount of the compound(s) is sufficient for increasing the effect of the prevention or treatment of Alzheimer's disease or for inhibiting any detrimental side effect of said administering step.
 5. The method according to claim 3, wherein the total amount of the compound(s) is 5 μg to 250 mg.
 6. The method according to claim 3, wherein the compound or mixture of compounds is selected from the group consisting of vitamin C, vitamin E, Q10, and combinations thereof.
 7. The method according to claim 3, wherein the compound or mixture of compounds is selected from the group consisting of tacrine and donepezil.
 8. The method according to claim 1, further comprising administering vitamin B₁₂ to the subject.
 9. A method of treating a subject having or suspected of having Alzheimer's disease comprising administering to the subject an amount, effective for the treatment of prevention of Azheimer's disease, of (a) phanquinone; and (b) a mixture of clioquinol and vitamin B₁₂, the amount of vitamin B₁₂ being effective to inhibit a detrimental side effect of clioquinol administration, said subject being in need of said treatment or prevention.
 10. The method according to claim 9, wherein the amount of clioquinol is 5 mg to 250 mg.
 11. The method according to claim 9, wherein the amount of vitamin B₁₂ is 5 μg to 2 mg.
 12. The method according to claim 3, wherein (a) phanquinone and (b) the compound(s) are present in a single pharmaceutical composition.
 13. The method according to claim 3, wherein (a) phanquinone and (b) the compound(s) are administered substantially simultaneously.
 14. The method according to claim 3, wherein (a) phanquinone and (b) the compound(s) are administered sequentially.
 15. The method according to claim 9, wherein clioquinol and vitamin B₁₂ are administered sequentially, phanquinone being administered together with clioquinol, vitamin B₁₂ or separately.
 16. The method according to claim 9, wherein clioquinol is administered in a first period, followed by a second period wherein vitamin B₁₂ is administered, phanquinone being administered together with clioquinol, vitamin B₁₂ or separately.
 17. The method according to claim 16, wherein the first period is one to three weeks and the second period is one to four weeks.
 18. The method according to any of claims 1-7, 8-10, 11 and 12-17, wherein the subject is human.
 19. The method according to claim 1 or 9, wherein phanquinone is administered for up to ten years.
 20. The method according to claim 1 or 9, wherein phanquinone is formulated for oral administration, parenteral administration or intradermal administration. 